Lettuce cultivar rio bravo

ABSTRACT

A lettuce cultivar, designated Rio Bravo, is disclosed. The invention relates to the seeds of lettuce cultivar Rio Bravo, to the plants of lettuce cultivar Rio Bravo and to methods for producing a lettuce plant by crossing the cultivar Rio Bravo with itself or another lettuce cultivar. The invention further relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic lettuce plants and plant parts produced by those methods. This invention also relates to lettuce cultivars or breeding cultivars and plant parts derived from lettuce cultivar Rio Bravo, to methods for producing other lettuce cultivars, lines or plant parts derived from lettuce cultivar Rio Bravo and to the lettuce plants, varieties, and their parts derived from the use of those methods. The invention further relates to hybrid lettuce seeds, plants, and plant parts produced by crossing cultivar Rio Bravo with another lettuce cultivar.

BACKGROUND OF THE INVENTION

The present invention relates to a leaf lettuce (Lactuca sativa L.)variety designated Rio Bravo. All publications cited in this applicationare herein incorporated by reference.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety or hybridan improved combination of desirable traits from the parental germplasm.These important traits may include increased head size and weight,higher seed yield, improved color, resistance to diseases and insects,tolerance to drought and heat, and better agronomic quality.

Practically speaking, all cultivated forms of lettuce belong to thehighly polymorphic species Lactuca sativa that is grown for its ediblehead and leaves. As a crop, lettuce is grown commercially whereverenvironmental conditions permit the production of an economically viableyield. Lettuce is the World's most popular salad. In the United States,the principal growing regions are California and Arizona which produceapproximately 329,700 acres out of a total annual acreage of more than333,300 acres (USDA (2005)). Fresh lettuce is available in the UnitedStates year-round although the greatest supply is from May throughOctober. For planting purposes, the lettuce season is typically dividedinto three categories (i.e., early, mid, and late), with the coastalareas planting from January to August, and the desert regions plantingfrom August to December. Fresh lettuce is consumed nearly exclusively asfresh, raw product and occasionally as a cooked vegetable.

Lactuca sativa is in the Cichoreae tribe of the Asteraceae (Compositae)family. Lettuce is related to chicory, sunflower, aster, dandelion,artichoke, and chrysanthemum. Sativa is one of about 300 species in thegenus Lactuca. There are seven different morphological types of lettuce.The crisphead group includes the iceberg and batavian types. Iceberglettuce has a large, firm head with a crisp texture and a white orcreamy yellow interior. The batavian lettuce predates the iceberg typeand has a smaller and less firm head. The butterhead group has a small,soft head with an almost oily texture. The romaine, also known as coslettuce, has elongated upright leaves forming a loose, loaf-shaped headand the outer leaves are usually dark green. Leaf lettuce comes in manyvarieties, none of which form a head, and include the green oak leafvariety. Latin lettuce looks like a cross between romaine andbutterhead. Stem lettuce has long, narrow leaves and thick, ediblestems. Oilseed lettuce is a type grown for its large seeds that arepressed to obtain oil. Latin lettuce, stem lettuce, and oilseed lettuceare seldom seen in the United States.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard, theoverall value of the advanced breeding lines, and the number ofsuccessful cultivars produced per unit of input (e.g., per year, perdollar expended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for at least three years. The best lines are candidatesfor new commercial cultivars. Those still deficient in a few traits areused as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from ten to twenty years from the time thefirst cross or selection is made. Therefore, development of newcultivars is a time-consuming process that requires precise forwardplanning, efficient use of resources, and a minimum of changes indirection.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of lettuce plant breeding is to develop new, unique, andsuperior lettuce cultivars. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing, and mutations. The breeder has no direct control at thecellular level. Therefore, two breeders will never develop the sameline, or even very similar lines, having the same lettuce traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under different geographical,climatic, and soil conditions, and further selections are then madeduring, and at the end of, the growing season. The cultivars that aredeveloped are unpredictable. This unpredictability is because thebreeder's selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same line twice by using the exactsame original parents and the same selection techniques. Thisunpredictability results in the expenditure of large research monies todevelop superior lettuce cultivars.

The development of commercial lettuce cultivars requires the developmentof lettuce varieties, the crossing of these varieties, and theevaluation of the crosses. Pedigree breeding and recurrent selectionbreeding methods are used to develop cultivars from breedingpopulations. Breeding programs combine desirable traits from two or morevarieties or various broad-based sources into breeding pools from whichcultivars are developed by selfing and selection of desired phenotypes.The new cultivars are crossed with other varieties and the hybrids fromthese crosses are evaluated to determine which have commercialpotential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁'s or by intercrossing two F₁'s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population. Then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror line that is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent areselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineswith each generation due to failure of some seeds to germinate or someplants to produce at least one seed. As a result, not all of the F₂plants originally sampled in the population will be represented by aprogeny when generation advance is completed.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length polymorphisms (AFLPs), Simple Sequence Repeats(SSRs—which are also referred to as Microsatellites), and SingleNucleotide Polymorphisms (SNPs).

Isozyme Electrophoresis and RFLPs have been widely used to determinegenetic composition. Shoemaker and Olsen (Molecular Linkage Map ofSoybean (Glycine max), pp. 6.131-6.138 in S. J. O'Brien (ed.) GeneticMaps: Locus Maps of Complex Genomes, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1993)) developed a molecular geneticlinkage map that consisted of 25 linkage groups with about 365 RFLP, 11RAPD, three classical markers, and four isozyme loci. See also,Shoemaker, R. C., RFLP Map of Soybean, pp. 299-309, in Phillips, R. L.and Vasil, I. K. (eds.), DNA-Based Markers in Plants, Kluwer AcademicPress, Dordrecht, the Netherlands (1994).

SSR technology is currently the most efficient and practical markertechnology; more marker loci can be routinely used and more alleles permarker locus can be found using SSRs in comparison to RFLPs. Forexample, Diwan and Cregan described a highly polymorphic microsatellitelocus in soybean with as many as 26 alleles. Diwan, N. and Cregan, P.B., Theor. Appl. Genet., 95:22-225 (1997). SNPs may also be used toidentify the unique genetic composition of the invention and progenyvarieties retaining that unique genetic composition. Various molecularmarker techniques may be used in combination to enhance overallresolution.

Molecular markers, which include markers identified through the use oftechniques such as Isozyme Electrophoresis, RFLPs, RAPDs, AP-PCR, DAF,SCARs, AFLPs, SSRs, and SNPs, may be used in plant breeding. One use ofmolecular markers is Quantitative Trait Loci (QTL) mapping. QTL mappingis the use of markers which are known to be closely linked to allelesthat have measurable effects on a quantitative trait. Selection in thebreeding process is based upon the accumulation of markers linked to thepositive effecting alleles and/or the elimination of the markers linkedto the negative effecting alleles from the plant's genome.

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select toward the genome of the recurrent parent and against themarkers of the donor parent. This procedure attempts to minimize theamount of genome from the donor parent that remains in the selectedplants. It can also be used to reduce the number of crosses back to therecurrent parent needed in a backcrossing program. The use of molecularmarkers in the selection process is often called genetic marker enhancedselection or marker-assisted selection. Molecular markers may also beused to identify and exclude certain sources of germplasm as parentalvarieties or ancestors of a plant by providing a means of trackinggenetic profiles through crosses.

Mutation breeding is another method of introducing new traits intolettuce varieties. Mutations that occur spontaneously or areartificially induced can be useful sources of variability for a plantbreeder. The goal of artificial mutagenesis is to increase the rate ofmutation for a desired characteristic. Mutation rates can be increasedby many different means including temperature, long-term seed storage,tissue culture conditions, radiation (such as X-rays, Gamma rays,neutrons, Beta radiation, or ultraviolet radiation), chemical mutagens(such as base analogs like 5-bromo-uracil), antibiotics, alkylatingagents (such as sulfur mustards, nitrogen mustards, epoxides,ethyleneamines, sulfates, sulfonates, sulfones, or lactones), azide,hydroxylamine, nitrous acid, or acridines. Once a desired trait isobserved through mutagenesis the trait may then be incorporated intoexisting germplasm by traditional breeding techniques. Details ofmutation breeding can be found in Principles of Cultivar Development byFehr, Macmillan Publishing Company (1993).

The production of double haploids can also be used for the developmentof homozygous varieties in a breeding program. Double haploids areproduced by the doubling of a set of chromosomes from a heterozygousplant to produce a completely homozygous individual. For example, seeWan, et al., Theor. Appl. Genet., 77:889-892 (1989).

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Principles of Plant Breeding, John Wiley and Son, pp.115-161 (1960); Allard (1960); Simmonds (1979); Sneep, et al. (1979);Fehr (1987); “Carrots and Related Vegetable Umbelliferae,” Rubatzky, V.E., et al. (1999).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts, as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Lettuce in general, and leaf lettuce in particular, is an important andvaluable vegetable crop. Thus, a continuing goal of lettuce plantbreeders is to develop stable, high yielding lettuce cultivars that areagronomically sound. To accomplish this goal, the lettuce breeder mustselect and develop lettuce plants with traits that result in superiorcultivars.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools, and methods which are meant to beexemplary and illustrative, not limiting in scope. In variousembodiments, one or more of the above-described problems have beenreduced or eliminated, while other embodiments are directed to otherimprovements.

According to the invention, there is provided a novel lettuce cultivardesignated Rio Bravo. This invention thus relates to the seeds oflettuce cultivar Rio Bravo, to the plants of lettuce cultivar Rio Bravo,and to methods for producing a lettuce plant produced by crossing thelettuce cultivar Rio Bravo with itself or another lettuce plant, tomethods for producing a lettuce plant containing in its genetic materialone or more transgenes, and to the transgenic lettuce plants produced bythat method. This invention also relates to methods for producing otherlettuce cultivars derived from lettuce cultivar Rio Bravo and to thelettuce cultivar derived by the use of those methods. This inventionfurther relates to hybrid lettuce seeds and plants produced by crossinglettuce cultivar Rio Bravo with another lettuce variety.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of lettuce cultivar Rio Bravo. The tissue culturewill preferably be capable of regenerating plants having essentially allof the physiological and morphological characteristics of the foregoinglettuce plant, and of regenerating plants having substantially the samegenotype as the foregoing lettuce plant. Preferably, the regenerablecells in such tissue cultures will be callus, protoplasts, meristematiccells, cotyledons, hypocotyl, leaves, pollen, embryos, roots, root tips,anthers, pistils, shoots, stems, petiole flowers, and seeds. Stillfurther, the present invention provides lettuce plants regenerated fromthe tissue cultures of the invention.

Another aspect of the invention is to provide methods for producingother lettuce plants derived from lettuce cultivar Rio Bravo. Lettucecultivars derived by the use of those methods are also part of theinvention.

The invention also relates to methods for producing a lettuce plantcontaining in its genetic material one or more transgenes and to thetransgenic lettuce plant produced by those methods.

In another aspect, the present invention provides for single geneconverted plants of Rio Bravo. The single transferred gene maypreferably be a dominant or recessive allele. Preferably, the singletransferred gene will confer such traits as male sterility, herbicideresistance, insect or pest resistance, modified fatty acid metabolism,modified carbohydrate metabolism, resistance for bacterial, fungal, orviral disease, male fertility, enhanced nutritional quality, andindustrial usage. The single gene may be a naturally occurring lettucegene or a transgene introduced through genetic engineering techniques.

The invention further provides methods for developing lettuce plants ina lettuce plant breeding program using plant breeding techniquesincluding recurrent selection, backcrossing, pedigree breeding,restriction fragment length polymorphism enhanced selection, geneticmarker enhanced selection, and transformation. Seeds, lettuce plants,and parts thereof, produced by such breeding methods are also part ofthe invention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference bystudy of the following descriptions.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. The allele is any of one or more alternative forms of a gene,all of which relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Big Vein virus. Big vein is a disease of lettuce caused by LettuceMirafiori Big Vein Virus which is transmitted by the fungus Olpidiumvirulentus, with vein clearing and leaf shrinkage resulting in plants ofpoor quality and reduced marketable value.

Bolting. The premature development of a flowering stalk, and subsequentseed, before a plant produces a food crop. Bolting is typically causedby late planting when temperatures are low enough to cause vernalizationof the plants.

Bremia lactucae. An Oomycete that causes downy mildew in lettuce incooler growing regions.

Core length. Length of the internal lettuce stem measured from the baseof the cut and trimmed head to the tip of the stem.

Corky root. A disease caused by the bacterium Sphingomonassuberifaciens, which causes the entire taproot to become brown, severelycracked, and non-functional.

Cotyledon. One of the first leaves of the embryo of a seed plant;typically one or more in monocotyledons, two in dicotyledons, and two ormore in gymnosperms.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

First water date. The date the seed first receives adequate moisture togerminate. This can and often does equal the planting date.

Gene. As used herein, “gene” refers to a segment of nucleic acid. A genecan be introduced into a genome of a species, whether from a differentspecies or from the same species, using transformation or variousbreeding methods.

Head diameter. Diameter of the cut and trimmed head, sliced vertically,and measured at the widest point perpendicular to the stem.

Head height. Height of the cut and trimmed head, sliced vertically, andmeasured from the base of the cut stem to the cap leaf.

Head weight. Weight of saleable lettuce head, cut and trimmed to marketspecifications.

Lettuce Mosaic virus. A disease that can cause a stunted, deformed, ormottled pattern in young lettuce and yellow, twisted, and deformedleaves in older lettuce.

Maturity date. Maturity refers to the stage when the plants are of fullsize or optimum weight, in marketable form or shape to be of commercialor economic value.

Nasonovia ribisnigri. A lettuce aphid that colonizes the innermostleaves of the lettuce plant, contaminating areas that cannot be treatedeasily with insecticides.

Quantitative Trait Loci. Quantitative Trait Loci (QTL) refers to geneticloci that control to some degree, numerically representable traits thatare usually continuously distributed.

Ratio of head height/diameter. Head height divided by the head diameteris an indication of the head shape; <1 is flattened, 1=round, and >1 ispointed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

RHS. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system. The chart may bepurchased from Royal Horticulture Society Enterprise Ltd., RHS Garden;Wisley, Woking; Surrey GU236QB, UK.

Single gene converted. Single gene converted or conversion plant refersto plants which are developed by a plant breeding technique calledbackcrossing or via genetic engineering wherein essentially all of thedesired morphological and physiological characteristics of a line arerecovered in addition to the single gene transferred into the line viathe backcrossing technique or via genetic engineering.

Tip burn. Means a browning of the edges or tips of lettuce leaves thatis a physiological response to a lack of calcium.

Tomato Bushy Stunt. A disease which causes stunting of growth, leafmottling, and deformed or absent fruit.

DETAILED DESCRIPTION OF THE INVENTION

Lettuce cultivar Rio Bravo is a lettuce variety suitable for productionin California and the Arizona desert of the United States. Additionally,lettuce cultivar Rio Bravo is highly resistant to Tomato Bushy StuntVirus (TBSV) and Downy Mildew. It is the result of numerous generationsof plant selections chosen for its type and disease resistances.

The cultivar has shown uniformity and stability for the traits, withinthe limits of environmental influence for the traits. It has beenself-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The line has been increased withcontinued observation for uniformity. No variant traits have beenobserved or are expected in cultivar Rio Bravo.

Lettuce cultivar Rio Bravo has the following morphologic and othercharacteristics, described in Table 1.

TABLE 1 VARIETY DESCRIPTION INFORMATION Plant: Type: Green RomaineMaturity date: 65 days (Summer), 115 days (winter) from first waterdate. Seed: Color: Black Light dormancy: Light not required Heatdormancy: Susceptible Cotyledon (to fourth leaf stage): Shape: BroadUndulation: Flat Anthocyanin distribution: Absent Rolling: AbsentCupping: Uncupped Reflexing: None Mature Leaves: Margin: Incision depth:Absent/Shallow Indentation: Shallowly Dentate Undulation of the apicalmargin: Absent/Slight Hue of green color of outer leaves: Dark GreenAnthocyanin distribution: Absent Glossiness: Dull Blistering: ModerateThickness: Thick Trichomes: Absent Plant at Market Stage Head shape:Slight V Shaped Head size class: Large Head weight (g): 720.7 Headfirmness: Moderate Core: Diameter at base of head (cm):  3.51 Coreheight from base of head  6.44 to apex (cm): Primary Regions ofAdaptation: Spring area: Salinas, California, Imperial, California, SanJoaquin, California, and Yuma, Arizona (United States) Summer area:Salinas, California, Santa Maria, California, and San Benito, California(United States) Autumn area: Yuma, Arizona, Imperial, California, andSalinas, California (United States) Winter area: Yuma, Arizona,Imperial, California, and Coachella, California (United States) Diseaseand Stress Reactions: Tomato Bushy Stunt Virus Highly resistant (TBSV):Big Vein: Intermediate Downy Mildew (Bremia lactucae): Highly resistantTipburn: Tolerant Heat: Intermediate Cold: Tolerant Brown Rib: ResistantPink Rib: Resistant Rusty Brown Discoloration: Resistant Internal RibNecrosis (Blackheart, Resistant Gray Rib, Gray Streak):

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct gene transfer method, suchas microprojectile-mediated delivery, DNA injection, electroporation,and the like. More preferably, expression vectors are introduced intoplant tissues by using either microprojectile-mediated delivery with abiolistic device or by using Agrobacterium-mediated transformation.Transformed plants obtained with the protoplasm of the invention areintended to be within the scope of this invention.

Further Embodiments of the Invention

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Any DNA sequences,whether from a different species or from the same species, which areintroduced into the genome using transformation or various breedingmethods, are referred to herein collectively as “transgenes.” Over thelast fifteen to twenty years, several methods for producing transgenicplants have been developed, and the present invention, in particularembodiments, also relates to transformed versions of the claimed line.

Nucleic acids or polynucleotides refer to RNA or DNA that is linear orbranched, single or double stranded, or a hybrid thereof. The term alsoencompasses RNA/DNA hybrids. These terms also encompass untranslatedsequence located at both the 3′ and 5′ ends of the coding region of thegene: at least about 1000 nucleotides of sequence upstream from the 5′end of the coding region and at least about 200 nucleotides of sequencedownstream from the 3′ end of the coding region of the gene. Less commonbases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine,and others can also be used for antisense, dsRNA, and ribozyme pairing.For example, polynucleotides that contain C-5 propyne analogues ofuridine and cytidine have been shown to bind RNA with high affinity andto be potent antisense inhibitors of gene expression. Othermodifications, such as modification to the phosphodiester backbone, orthe 2′-hydroxy in the ribose sugar group of the RNA can also be made.The antisense polynucleotides and ribozymes can consist entirely ofribonucleotides, or can contain mixed ribonucleotides anddeoxyribonucleotides. The polynucleotides of the invention may beproduced by any means, including genomic preparations, cDNApreparations, in vitro synthesis, RT-PCR, and in vitro or in vivotranscription.

Plant transformation involves the construction of an expression vectorthat will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid, and can beused alone or in combination with other plasmids, to provide transformedlettuce plants using transformation methods as described below toincorporate transgenes into the genetic material of the lettuceplant(s).

Expression Vectors for Lettuce Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (for example, a promoter) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or an herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley, et al., PNAS, 80:4803 (1983).Another commonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin. Vanden Elzen, et al., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford, et al., Plant Physiol.,86:1216 (1988); Jones, et al., Mol. Gen. Genet., 210:86 (1987); Svab, etal., Plant Mol. Biol., 14:197 (1990); Hille, et al., Plant Mol. Biol.,7:171 (1986). Other selectable marker genes confer resistance toherbicides such as glyphosate, glufosinate, or bromoxynil. Comai, etal., Nature, 317:741-744 (1985); Gordon-Kamm, et al., Plant Cell,2:603-618 (1990); and Stalker, et al., Science, 242:419-423 (1988).

Selectable marker genes for plant transformation that are not ofbacterial origin include, for example, mouse dihydrofolate reductase,plant 5-enolpyruvylshikimate-3-phosphate synthase, and plantacetolactate synthase. Eichholtz, et al., Somatic Cell Mol. Genet.,13:67 (1987); Shah, et al., Science, 233:478 (1986); and Charest, etal., Plant Cell Rep., 8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include α-glucuronidase (GUS),α-galactosidase, luciferase and chloramphenicol, acetyltransferase.Jefferson, R. A., Plant Mol. Biol., 5:387 (1987); Teeri, et al., EMBOJ., 8:343 (1989); Koncz, et al., PNAS, 84:131 (1987); and DeBlock, etal., EMBO J., 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissues are available. Molecular Probes,Publication 2908, IMAGENE GREEN, pp. 1-4 (1993) and Naleway, et al., J.Cell Biol., 115:151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds, and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie, et al., Science, 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Expression Vectors for Lettuce Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element (for example, a promoter).Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific.” A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters:

An inducible promoter is operably linked to a gene for expression inlettuce. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in lettuce. With an inducible promoter, therate of transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward,et al., Plant Mol. Biol., 22:361-366 (1993). Exemplary induciblepromoters include, but are not limited to, that from the ACEI systemwhich responds to copper (Meft, et al., PNAS, 90:4567-4571 (1993)); In2gene from maize which responds to benzenesulfonamide herbicide safeners(Hershey, et al., Mol. Gen. Genet., 227:229-237 (1991) and Gatz, et al.,Mol. Gen. Genet., 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz, etal., Mol. Gen. Genet., 227:229-237 (1991)). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena,et al., PNAS, 88:0421 (1991).

B. Constitutive Promoters:

A constitutive promoter is operably linked to a gene for expression inlettuce or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in lettuce.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell, et al., Nature, 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy, et al., Plant Cell, 2:163-171 (1990));ubiquitin (Christensen, et al., Plant Mol. Biol., 12:619-632 (1989) andChristensen, et al., Plant Mol. Biol., 18:675-689 (1992)); pEMU (Last,et al., Theor. Appl. Genet., 81:581-588 (1991)); MAS (Velten, et al.,EMBO J., 3:2723-2730 (1984)) and maize H3 histone (Lepetit, et al., Mol.Gen. Genet., 231:276-285 (1992) and Atanassova, et al., Plant J., 2(3):291-300 (1992)). The ALS promoter, Xba1/Nco1 fragment 5′ to theBrassica napus ALS3 structural gene (or a nucleotide sequence similarityto said Xba1/Nco1 fragment), represents a particularly usefulconstitutive promoter. See PCT Application No. WO 96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters:

A tissue-specific promoter is operably linked to a gene for expressionin lettuce. Optionally, the tissue-specific promoter is operably linkedto a nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in lettuce. Plants transformed with agene of interest operably linked to a tissue-specific promoter producethe protein product of the transgene exclusively, or preferentially, ina specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai, et al., Science, 23:476-482(1983) and Sengupta-Gopalan, et al., PNAS, 82:3320-3324 (1985)); aleaf-specific and light-induced promoter such as that from cab orrubisco (Simpson, et al., EMBO J., 4(11):2723-2729 (1985) and Timko, etal., Nature, 318:579-582 (1985)); an anther-specific promoter such asthat from LAT52 (Twell, et al., Mol. Gen. Genet., 217:240-245 (1989)); apollen-specific promoter such as that from Zm13 (Guerrero, et al., Mol.Gen. Genet., 244:161-168 (1993)) or a microspore-preferred promoter suchas that from apg (Twell, et al., Sex. Plant Reprod., 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall, ormitochondrion, or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker, et al., Plant Mol. Biol., 20:49 (1992); Close, P. S.,Master's Thesis, Iowa State University (1993); Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley,” Plant Mol. Biol., 9:3-17 (1987); Lerner, et al., PlantPhysiol., 91:124-129 (1989); Fontes, et al., Plant Cell, 3:483-496(1991); Matsuoka, et al., PNAS, 88:834 (1991); Gould, et al., J. Cell.Biol., 108:1657 (1989); Creissen, et al., Plant J., 2:129 (1991);Kalderon, et al., A short amino acid sequence able to specify nuclearlocation, Cell, 39:499-509 (1984); and Steifel, et al., Expression of amaize cell wall hydroxyproline-rich glycoprotein gene in early leaf androot vascular differentiation, Plant Cell, 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem., 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is lettuce. In anotherpreferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR, and SSR analysis, which identifies the approximatechromosomal location of the integrated DNA molecule. For exemplarymethodologies in this regard, see Methods in Plant Molecular Biology andBiotechnology, Glick and Thompson Eds., 269:284, CRC Press, Boca Raton(1993). Map information concerning chromosomal location is useful forproprietary protection of a subject transgenic plant. If unauthorizedpropagation is undertaken and crosses made with other germplasm, the mapof the integration region can be compared to similar maps for suspectplants, to determine if the latter have a common parentage with thesubject plant. Map comparisons would involve hybridizations, RFLP, PCR,SSR, and sequencing, all of which are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

A. Genes That Confer Resistance to Pests or Disease and That Encode:

1. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant line can be transformed with a clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Jones, et al., Science, 266:789(1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfulvum); Martin, et al., Science, 262:1432 (1993) (tomato Pto gene forresistance to Pseudomonas syringae pv. tomato encodes a protein kinase);and Mindrinos, et al., Cell, 78:1089 (1994) (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae).

2. A Bacillus thuringiensis protein, a derivative thereof, or asynthetic polypeptide modeled thereon. See, for example, Geiser, et al.,Gene, 48:109 (1986), who disclose the cloning and nucleotide sequence ofa Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Nos. 40098, 67136, 31995, and31998.

3. A lectin. See, for example, the disclosure by Van Damme, et al.,Plant Mol. Biol., 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

4. A vitamin-binding protein such as avidin. See PCT Application No. US93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

5. An enzyme inhibitor, for example, a protease or proteinase inhibitor,or an amylase inhibitor. See, for example, Abe, et al., J. Biol. Chem.,262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor); Huub, et al., Plant Mol. Biol., 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I); and Sumitani,et al., Biosci. Biotech. Biochem., 57:1243 (1993) (nucleotide sequenceof Streptomyces nitrosporeus α-amylase inhibitor).

6. An insect-specific hormone or pheromone, such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock, et al., Nature, 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

7. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem., 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor) and Pratt, etal., Biochem. Biophys. Res. Comm., 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also, U.S. Pat. No. 5,266,317 toTomalski, et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

8. An insect-specific venom produced in nature, by a snake, a wasp, etc.For example, see Pang, et al., Gene, 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

9. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative,or another non-protein molecule with insecticidal activity.

10. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase, and a glucanase, whether natural or synthetic. See PCTApplication No. WO 93/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase gene. DNA molecules whichcontain chitinase-encoding sequences can be obtained, for example, fromthe ATCC under Accession Nos. 39637 and 67152. See also, Kramer, et al.,Insect Biochem. Mol. Biol., 23:691 (1993), who teach the nucleotidesequence of a cDNA encoding tobacco hornworm chitinase, and Kawalleck,et al., Plant Mol. Biol., 21:673 (1993), who provide the nucleotidesequence of the parsley ubi4-2 polyubiquitin gene.

11. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella, et al., Plant Mol. Biol., 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess,et al., Plant Physiol., 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

12. A hydrophobic moment peptide. See PCT Application No. WO 95/16776(disclosure of peptide derivatives of tachyplesin which inhibit fungalplant pathogens) and PCT Application No. WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

13. A membrane permease, a channel former, or a channel blocker. Forexample, see the disclosure of Jaynes, et al., Plant Sci., 89:43 (1993),of heterologous expression of a cecropin-β, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

14. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy, et al., Ann. Rev. Phytopathol.,28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus, and tobacco mosaic virus. Id.

15. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. SeeTaylor, et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions, Edinburgh, Scotland (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

16. A virus-specific antibody. See, for example, Tavladoraki, et al.,Nature, 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

17. A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient released by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb, et al., Bio/technology,10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubart,et al., Plant J, 2:367 (1992).

18. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann, et al., Bio/technology, 10:305 (1992), have shownthat transgenic plants expressing the barley ribosome-inactivating genehave an increased resistance to fungal disease.

19. A lettuce mosaic potyvirus (LMV) coat protein gene introduced intoLactuca sativa in order to increase its resistance to LMV infection. SeeDinant, et al., Mol. Breeding, 3:1, 75-86 (1997).

Any of the above listed disease or pest resistance genes (1-19) can beintroduced into the claimed lettuce cultivar through a variety of meansincluding but not limited to transformation and crossing.

B. Genes That Confer Resistance to an Herbicide:

1. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee, etal., EMBO J., 7:1241 (1988) and Miki, et al., Theor. Appl. Genet.,80:449 (1990), respectively.

2. Glyphosate (resistance conferred by mutant5-enolpyruvlshikimate-3-phosphate synthase (EPSPS) and aroA genes,respectively) and other phosphono compounds, such as glufosinate(phosphinothricin acetyl transferase (PAT), dicamba and Streptomyceshygroscopicus phosphinothricin-acetyl transferase PAT bar genes), andpyridinoxy or phenoxy proprionic acids and cyclohexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah, et al., which discloses the nucleotide sequence of a form of EPSPSwhich can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC Accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. See also, Umaballava-Mobapathie in TransgenicResearch, 8:1, 33-44 (1999) that discloses Lactuca sativa resistant toglufosinate. European Patent Application No. 0 333 033 to Kumada, etal., and U.S. Pat. No. 4,975,374 to Goodman, et al., disclose nucleotidesequences of glutamine synthetase genes which confer resistance toherbicides, such as L-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in EuropeanApplication No. 0 242 246 to Leemans, et al. DeGreet et al.,Bio/technology, 7:61 (1989), describe the production of transgenicplants that express chimeric bar genes coding for phosphinothricinacetyl transferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop, are the Acc1-S1, Acc1-S2, and Acc1-S3 genes described byMarshall, et al., Theor. Appl. Genet., 83:435 (1992).

3. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla, et al.,Plant Cell, 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes, et al., Biochem. J.,285:173 (1992).

4. Acetohydroxy acid synthase, which has been found to make plants thatexpress this enzyme resistant to multiple types of herbicides, has beenintroduced into a variety of plants. See Hattori, et al., Mol. Gen.Genet., 246:419 (1995). Other genes that confer tolerance to herbicidesinclude a gene encoding a chimeric protein of rat cytochrome P4507A1 andyeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., PlantPhysiol., 106:17 (1994)), genes for glutathione reductase and superoxidedismutase (Aono, et al., Plant Cell Physiol., 36:1687 (1995)), and genesfor various phosphotransferases (Datta, et al., Plant Mol. Biol., 20:619(1992)).

5. Protoporphyrinogen oxidase (protox) is necessary for the productionof chlorophyll, which is necessary for all plant survival. The protoxenzyme serves as the target for a variety of herbicidal compounds. Theseherbicides also inhibit growth of all the different species of plantspresent, causing their total destruction. The development of plantscontaining altered protox activity which are resistant to theseherbicides are described in U.S. Pat. Nos. 6,288,306, 6,282,837,5,767,373, and International Publication WO 01/12825.

Any of the above listed herbicide genes (1-5) can be introduced into theclaimed lettuce cultivar through a variety of means including, but notlimited to, transformation and crossing.

C. Genes that Confer or Contribute to a Value-Added Trait, Such as:

1. Increased iron content of the lettuce, for example, by introducinginto a plant a soybean ferritin gene as described in Goto, et al., ActaHorticulturae., 521, 101-109 (2000).

2. Decreased nitrate content of leaves, for example, by introducing intoa lettuce a gene coding for a nitrate reductase. See, for example,Curtis, et al., Plant Cell Rep., 18:11, 889-896 (1999).

3. Increased sweetness of the lettuce by introducing a gene coding formonellin that elicits a flavor 100,000 times sweeter than sugar on amolar basis. See Penarrubia, et al., Bio/technology, 10:561-564 (1992).

4. Modified fatty acid metabolism, for example, by introducing into aplant an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon, et al., PNAS, 89:2625 (1992).

5. Modified carbohydrate composition effected, for example, byintroducing into plants a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza, et al., J. Bacteriol., 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene); Steinmetz, et al., Mol. Gen. Genet., 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene);Pen, et al., Bio/technology, 10:292 (1992) (production of transgenicplants that express Bacillus lichenifonnis α-amylase); Elliot, et al.,Plant Mol. Biol., 21:515 (1993) (nucleotide sequences of tomatoinvertase genes); Søgaard, et al., J. Biol. Chem., 268:22480 (1993)(site-directed mutagenesis of barley α-amylase gene); and Fisher, etal., Plant Physiol., 102:1045 (1993) (maize endosperm starch branchingenzyme II).

D. Genes that Control Male-Sterility:

1. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN—Ac-PPT. See International Publication WO 01/29237.

2. Introduction of various stamen-specific promoters. See InternationalPublications WO 92/13956 and WO 92/13957.

3. Introduction of the barnase and the barstar genes. See Paul, et al.,Plant Mol. Biol., 19:611-622 (1992).

Methods for Lettuce Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki, et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glickand Thompson Eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). Inaddition, expression vectors and in vitro culture methods for plant cellor tissue transformation and regeneration of plants are available. See,for example, Gruber, et al., “Vectors for Plant Transformation” inMethods in Plant Molecular Biology and Biotechnology, Glick and ThompsonEds., CRC Press, Inc., Boca Raton, pp. 89-119 (1993).

A. Agrobacterium-Mediated Transformation:

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch, et al., Science, 227:1229 (1985); Curtis, et al., Journal ofExperimental Botany, 45:279, 1441-1449 (1994); Torres, et al., PlantCell Tissue and Organ Culture, 34:3, 279-285 (1993); and Dinant, et al.,Molecular Breeding, 3:1, 75-86 (1997). A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci., 10:1(1991). Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber, et al.,supra, Miki, et al., supra, and Moloney, et al., Plant Cell Rep., 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer:

Several methods of plant transformation collectively referred to asdirect gene transfer have been developed as an alternative toAgrobacterium-mediated transformation. A generally applicable method ofplant transformation is microprojectile-mediated transformation whereinDNA is carried on the surface of microprojectiles measuring 1 μm to 4μm. The expression vector is introduced into plant tissues with abiolistic device that accelerates the microprojectiles to speeds of 300m/s to 600 m/s which is sufficient to penetrate plant cell walls andmembranes. Russell, D. R., et al., Plant Cell Rep., 12 (3, Jan.),165-169 (1993); Aragao, F. J. L., et al., Plant Mol. Biol., 20 (2,Oct.), 357-359 (1992); Aragao, F. J. L., et al., Plant Cell Rep., 12 (9,July), 483-490 (1993); Aragao, Theor. Appl. Genet., 93:142-150 (1996);Kim, J., Minamikawa, T., Plant Sci., 117:131-138 (1996); Sanford, etal., Part. Sci. Technol., 5:27 (1987); Sanford, J. C., Trends Biotech.,6:299 (1988); Klein, et al., Bio/technology, 6:559-563 (1988); Sanford,J. C., Physiol. Plant, 7:206 (1990); Klein, et al., Bio/technology,10:268 (1992).

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang, et al., Bio/technology, 9:996 (1991).Alternatively, liposome and spheroplast fusion have been used tointroduce expression vectors into plants. Deshayes, et al., EMBO J.,4:2731 (1985) and Christou, et al., PNAS, 84:3962 (1987). Direct uptakeof DNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol, orpoly-L-ornithine has also been reported. Hain, et al., Mol. Gen. Genet.,199:161 (1985) and Draper, et al., Plant Cell Physiol., 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Saker, M., Kuhne, T., Biologia Plantarum, 40(4):507-514(1997/98); Donn, et al., In Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p. 53 (1990); D'Halluin, etal., Plant Cell, 4:1495-1505 (1992); and Spencer, et al., Plant Mol.Biol., 24:51-61 (1994). See also Chupean, et al., Bio/technology, 7:5,503-508 (1989).

Following transformation of lettuce target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic line. The transgenic line could then be crossedwith another (non-transformed or transformed) line in order to produce anew transgenic lettuce line. Alternatively, a genetic trait which hasbeen engineered into a particular lettuce cultivar using the foregoingtransformation techniques could be introduced into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite inbred line into anelite inbred line, or from an inbred line containing a foreign gene inits genome into an inbred line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

Gene Conversions

When the term “lettuce plant” is used in the context of the presentinvention, this also includes any gene conversions of that variety. Theterm “gene converted plant” as used herein refers to those lettuceplants which are developed by backcrossing, genetic engineering, ormutation, wherein essentially all of the desired morphological andphysiological characteristics of a variety are recovered in addition tothe one or more genes transferred into the variety via the backcrossingtechnique, genetic engineering, or mutation. Backcrossing methods can beused with the present invention to improve or introduce a characteristicinto the variety. The term “backcrossing” as used herein refers to therepeated crossing of a hybrid progeny back to the recurrent parent,i.e., backcrossing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more times to therecurrent parent. The parental lettuce plant which contributes the genefor the desired characteristic is termed the “nonrecurrent” or “donorparent.” This terminology refers to the fact that the nonrecurrentparent is used one time in the backcross protocol and therefore does notrecur. The parental lettuce plant to which the gene or genes from thenonrecurrent parent are transferred is known as the recurrent parent asit is used for several rounds in the backcrossing protocol. Poehlman &Sleper (1994) and Fehr (1993). In a typical backcross protocol, theoriginal variety of interest (recurrent parent) is crossed to a secondvariety (nonrecurrent parent) that carries the gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until alettuce plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the transferredgene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a trait or characteristic in the original line.To accomplish this, a gene of the recurrent cultivar is modified orsubstituted with the desired gene from the nonrecurrent parent, whileretaining essentially all of the rest of the desired genetic, andtherefore the desired physiological and morphological, constitution ofthe original line. The choice of the particular nonrecurrent parent willdepend on the purpose of the backcross. One of the major purposes is toadd some commercially desirable, agronomically important trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered to determine an appropriate testing protocol.Although backcrossing methods are simplified when the characteristicbeing transferred is a dominant allele, a recessive allele may also betransferred. In this instance it may be necessary to introduce a test ofthe progeny to determine if the desired characteristic has beensuccessfully transferred.

Many gene traits have been identified that are not regularly selected inthe development of a new line but that can be improved by backcrossingtechniques. Gene traits may or may not be transgenic. Examples of thesetraits include, but are not limited to, male sterility, modified fattyacid metabolism, modified carbohydrate metabolism, herbicide resistance,resistance for bacterial, fungal, or viral disease, insect resistance,enhanced nutritional quality, industrial usage, yield stability, andyield enhancement. These genes are generally inherited through thenucleus. Several of these gene traits are described in U.S. Pat. Nos.5,777,196, 5,948,957, and 5,969,212, the disclosures of which arespecifically hereby incorporated by reference.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of lettuce andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Teng, et al., HortScience, 27:9,1030-1032 (1992); Teng, et al., HortScience, 28:6, 669-1671 (1993);Zhang, et al., Journal of Genetics and Breeding, 46:3, 287-290 (1992);Webb, et al., Plant Cell Tissue and Organ Culture, 38:1, 77-79 (1994);Curtis, et al., Journal of Experimental Botany, 45:279, 1441-1449(1994); Nagata, et al., Journal for the American Society forHorticultural Science, 125:6, 669-672 (2000); and Ibrahim, et al., PlantCell Tissue and Organ Culture, 28(2), 139-145 (1992). It is clear fromthe literature that the state of the art is such that these methods ofobtaining plants are routinely used and have a very high rate ofsuccess. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce lettuce plants having thephysiological and morphological characteristics of variety Rio Bravo.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, meristematic cells, andplant cells that can generate tissue culture that are intact in plantsor parts of plants, such as leaves, pollen, embryos, roots, root tips,anthers, pistils, flowers, seeds, petioles, suckers, and the like. Meansfor preparing and maintaining plant tissue culture are well known in theart. By way of example, a tissue culture comprising organs has been usedto produce regenerated plants. U.S. Pat. Nos. 5,959,185, 5,973,234, and5,977,445 describe certain techniques, the disclosures of which areincorporated herein by reference.

Additional Breeding Methods

This invention also is directed to methods for producing a lettuce plantby crossing a first parent lettuce plant with a second parent lettuceplant wherein the first or second parent lettuce plant is a lettuceplant of cultivar Rio Bravo. Further, both first and second parentlettuce plants can come from lettuce cultivar Rio Bravo. Thus, any suchmethods using lettuce cultivar Rio Bravo are part of this invention:selfing, backcrosses, hybrid production, crosses to populations, and thelike. All plants produced using lettuce cultivar Rio Bravo as at leastone parent are within the scope of this invention, including thosedeveloped from cultivars derived from lettuce cultivar Rio Bravo.Advantageously, this lettuce cultivar could be used in crosses withother, different, lettuce plants to produce the first generation (F₁)lettuce hybrid seeds and plants with superior characteristics. Thecultivar of the invention can also be used for transformation whereexogenous genes are introduced and expressed by the cultivar of theinvention. Genetic variants created either through traditional breedingmethods using lettuce cultivar Rio Bravo or through transformation ofcultivar Rio Bravo by any of a number of protocols known to those ofskill in the art are intended to be within the scope of this invention.

The following describes breeding methods that may be used with lettucecultivar Rio Bravo in the development of further lettuce plants. Onesuch embodiment is a method for developing cultivar Rio Bravo progenylettuce plants in a lettuce plant breeding program comprising: obtainingthe lettuce plant, or a part thereof, of cultivar Rio Bravo, utilizingsaid plant or plant part as a source of breeding material, and selectinga lettuce cultivar Rio Bravo progeny plant with molecular markers incommon with cultivar Rio Bravo and/or with morphological and/orphysiological characteristics selected from the characteristics listedin Table 1. Breeding steps that may be used in the lettuce plantbreeding program include pedigree breeding, backcrossing, mutationbreeding, and recurrent selection. In conjunction with these steps,techniques such as RFLP-enhanced selection, genetic marker enhancedselection (for example, SSR markers), and the making of double haploidsmay be utilized.

Another method involves producing a population of lettuce cultivar RioBravo progeny lettuce plants, comprising crossing cultivar Rio Bravowith another lettuce plant, thereby producing a population of lettuceplants, which, on average, derive 50% of their alleles from lettucecultivar Rio Bravo. A plant of this population may be selected andrepeatedly selfed or sibbed with a lettuce cultivar resulting from thesesuccessive filial generations. One embodiment of this invention is thelettuce cultivar produced by this method and that has obtained at least50% of its alleles from lettuce cultivar Rio Bravo.

One of ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is nosignificant difference between the two traits expressed by thosevarieties. For example, see Fehr and Walt, Principles of CultivarDevelopment, pp. 261-286 (1987). Thus the invention includes lettucecultivar Rio Bravo progeny lettuce plants comprising a combination of atleast two cultivar Rio Bravo traits selected from the group consistingof those listed in Table 1 or the cultivar Rio Bravo combination oftraits listed in the Summary of the Invention, so that said progenylettuce plant is not significantly different for said traits thanlettuce cultivar Rio Bravo as determined at the 5% significance levelwhen grown in the same environmental conditions. Using techniquesdescribed herein, molecular markers may be used to identify said progenyplant as a lettuce cultivar Rio Bravo progeny plant. Mean trait valuesmay be used to determine whether trait differences are significant, andpreferably the traits are measured on plants grown under the sameenvironmental conditions. Once such a variety is developed, its value issubstantial since it is important to advance the germplasm base as awhole in order to maintain or improve traits such as yield, diseaseresistance, pest resistance, and plant performance in extremeenvironmental conditions.

Progeny of lettuce cultivar Rio Bravo may also be characterized throughtheir filial relationship with lettuce cultivar Rio Bravo, as forexample, being within a certain number of breeding crosses of lettucecultivar Rio Bravo. A breeding cross is a cross made to introduce newgenetics into the progeny, and is distinguished from a cross, such as aself or a sib cross, made to select among existing genetic alleles. Thelower the number of breeding crosses in the pedigree, the closer therelationship between lettuce cultivar Rio Bravo and its progeny. Forexample, progeny produced by the methods described herein may be within1, 2, 3, 4, or 5 breeding crosses of lettuce cultivar Rio Bravo.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which lettuce plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as leaves, pollen, embryos,cotyledons, hypocotyl, roots, root tips, anthers, pistils, flowers,ovules, seeds, stems, and the like.

Tables

Table 2 compares the length of the cotyledon leaf in millimeters of 20day old seedlings of leaf lettuce cultivar Rio Bravo with commerciallettuce cultivars Green Thunder and True Heart. Data were taken in 2010in SALINAS, CALIFORNIA on 20 plants of each variety. Column 1 shows thecotyledon length for Rio Bravo, column 2 shows the cotyledon length forGreen Thunder, and column 3 shows the cotyledon length for True Heart.

TABLE 2 Rio Bravo Green Thunder True Heart 21 18 19 17 20 17 19 21 20 1920 18 19 21 21 20 20 18 18 19 19 18 18 20 19 22 20 18 20 19 18 21 22 1920 22 20 20 18 21 20 18 18 21 16 20 15 18 18 22 19 21 18 18 18 20 18 1720 19 Average 18.9 19.8 18.95

Table 3 compares the width of the cotyledon leaf in millimeters of 20day old seedlings of leaf lettuce cultivar Rio Bravo with commerciallettuce cultivars Green Thunder and True Heart. Data were taken in 2010in SALINAS, CALIFORNIA on 20 plants of each variety. Column 1 lists thecotyledon width for Rio Bravo, column 2 lists the cotyledon width forGreen Thunder, and column 3 lists the cotyledon width for True Heart.

TABLE 3 Rio Bravo Green Thunder True Heart 10 10 9 10 10 9 10 10 10 1010 9 10 10 11 9 9 9 10 9 9 10 9 9 8 11 11 9 9 10 10 11 10 11 9 11 10 109 10 9 10 10 10 8 8 8 10 10 10 10 10 9 9 9 10 9 9 10 9 Average 9.65 9.659.55

Table 4 compares the cotyledon leaf index (calculated by dividing thecotyledon leaf length by the cotyledon leaf width) in 20 day oldseedlings of leaf lettuce cultivar Rio Bravo with commercial lettucecultivars Green Thunder and True Heart. Data were taken in 2010 inSALINAS, CALIFORNIA on 20 plants of each variety. Column 1 lists thecotyledon index for Rio Bravo, column 2 lists the cotyledon index forGreen Thunder, and column 3 lists the cotyledon index for True Heart.

TABLE 4 Rio Bravo Green Thunder True Heart 2.1 1.8 2.1 1.7 2.0 1.9 1.92.1 2.0 1.9 2.0 2.0 1.9 2.1 1.9 2.2 2.2 2.0 1.8 2.1 2.1 1.8 2.0 2.2 2.42.0 1.8 2.0 2.2 1.9 1.8 1.9 2.2 1.7 2.2 2.0 2.0 2.0 2.0 2.1 2.2 1.8 1.82.1 2.0 2.5 1.9 1.8 1.8 2.2 1.9 2.1 2.0 2.0 2.0 2.0 2.0 1.9 2.0 2.1Average 1.9707 2.0542 1.9886

Table 5 compares the length of the 4^(th) true leaf measured incentimeters of 20 day old seedlings of leaf lettuce cultivar Rio Bravowith commercial lettuce cultivars Green Thunder and True Heart. Datawere taken in 2010 in SALINAS, CALIFORNIA on 20 plants of each variety.Column 1 shows the length of the 4^(th) true leaf for Rio Bravo, column2 shows the length of the 4^(th) true leaf for Green Thunder, and column3 shows the length of the 4^(th) true leaf for True Heart. The sum,average, and variance of the measurements for each variety is shown inthe last three rows.

TABLE 5 Rio Green Bravo Thunder True Heart 4.8 5.3 4.4 4.2 4.5 5.2 3.44.5 5.0 3.8 3.8 5.2 5.2 4.7 5.4 6.3 4.2 4.5 5.5 5.0 5.6 4.8 5.5 5.2 6.54.5 4.5 5.5 4.5 5.2 5.5 4.2 4.8 5.8 4.0 4.2 5.6 3.9 5.2 6.2 4.3 5.5 5.04.5 4.6 4.0 5.0 3.8 5.5 3.7 5.2 5.4 3.8 4.2 4.4 3.2 5.0 5.0 4.6 3.2 Sum102.4 87.7 95.9 Average 5.12 4.385 4.495 Variance 0.7038 0.3192 0.3784

Table 6 shows the results of a single-factor ANOVA testing for thelength of the 4^(th) true leaf between varieties, and within thevarieties, Rio Bravo, Green Thunder, and True Heart from data in Table5. Column 1 shows the source of variation, column 2 shows the sum ofsquares, column 3 shows the degrees of freedom, column 4 shows the meansquare, column 5 shows the F-value, column 6 shows the P-value, andcolumn 7 shows the F critical value. ANOVA results indicate asignificant difference in the length of the 4^(th) true leaf between thevarieties at 20 days old.

TABLE 6 Source of Sum of Degrees of Mean Variation Squares FreedomSquare F-value P-value F crit Between 5.4263 2 2.7132 5.8080 0.00513.1588 Varieties Within 26.627 57 0.4671 Varieties Total 32.053 59

Table 7 compares the width of the 4^(th) true leaf measured inmillimeters of 20 day old seedlings of leaf lettuce cultivar Rio Bravowith commercial lettuce cultivars Green Thunder and True Heart. Datawere taken in 2010 in SALINAS, CALIFORNIA on 20 plants of each variety.Column 1 shows the width of the 4^(th) true leaf for Rio Bravo, column 2shows the width of the 4^(th) true leaf for Green Thunder, and column 3shows the width of the 4^(th) true leaf for True Heart. The sum,average, and variance of the measurements for each variety is shown inthe last three rows.

TABLE 7 Rio Green Bravo Thunder True Heart 2.4 2.5 2.2 2.4 2.2 2.8 1.82.5 2.6 2.2 2.0 2.6 2.8 2.5 2.6 3.2 2.2 2.3 2.8 2.7 2.6 2.6 2.8 2.6 3.22.5 2.2 2.7 2.4 2.8 3.0 2.1 2.3 3.1 2.0 2.2 3.0 2.0 2.8 3.0 2.2 2.2 2.42.0 2.4 2.1 2.5 2.0 3.0 1.8 2.8 3.0 1.9 2.2 2.2 1.8 2.4 2.6 2.1 1.6 Sum53.5 44.7 48.5 Average 2.6750 2.2350 2.4100 Variance 0.1620 0.08770.0978

Table 8 shows the results of a single-factor ANOVA testing for the widthof the 4^(th) true leaf between varieties, and within the varieties, RioBravo, Green Thunder, and True Heart from data in Table 7. Column 1shows the source of variation, column 2 shows the sum of squares, column3 shows the degrees of freedom, column 4 shows the mean square, column 5shows the F-value, column 6 shows the P-value, and column 7 shows the Fcritical value. ANOVA results indicate a significant difference in thewidth of the 4^(th) true leaf between the varieties at 20 days old.

TABLE 8 Source of Sum of Degrees of Mean Variation Squares FreedomSquare F-value P-value F crit Between 1.963 2 0.9815 8.4753 0.00063.1588 Varieties Within 6.601 57 0.1158 Varieties Total 8.564 59

Table 9 compares the 4^(th) leaf index (calculated by dividing the4^(th) leaf length by the 4^(th) leaf width) of 20 day old seedlings ofleaf lettuce cultivar Rio Bravo with commercial lettuce cultivars GreenThunder and True Heart. Data were taken in 2010 in SALINAS, CALIFORNIAon 20 plants of each variety. Column 1 shows the 4^(th) leaf index forRio Bravo, column 2 shows the 4^(th) leaf index for Green Thunder, andcolumn 3 shows the 4^(th) leaf index for True Heart.

TABLE 9 Rio Bravo Green Thunder True Heart 2.0 2.1 2.0 1.8 2.0 1.9 1.91.8 1.9 1.7 1.9 2.0 1.9 1.9 2.1 2.0 1.9 2.0 2.0 1.9 2.2 1.8 2.0 2.0 2.01.8 2.0 2.0 1.9 1.9 1.8 2.0 2.1 1.9 2.0 1.9 1.9 2.0 1.9 2.1 2.0 2.5 2.12.3 1.9 1.9 2.0 1.9 1.8 2.1 1.9 1.8 2.0 1.9 2.0 1.8 2.1 1.9 2.2 2.0Average 1.9126 1.9662 1.9945

Tables 10 through 12 show the plant weight in grams at harvest maturityfor Rio Bravo, (Table 10) Green Thunder, (Table 11) and True Heart(Table 12). The data were taken in 2008 TO 2010 at seven trials inWELLTON AND YUMA, ARIZONA, AND BARD AND SALINAS, CALIFORNIA for 20samples of each variety.

TABLE 10 Rio Bravo 1 2 3 4 5 6 7 354 659 379 870 1035 896 1040 425 644487 875 860 683 940 380 669 606 868 743 596 990 308 597 525 870 896 759990 401 493 613 1042 931 587 1020 370 640 568 949 687 703 1040 498 608497 756 936 752 1080 471 606 605 703 870 817 1140 356 621 551 1043 1044782 890 250 631 582 683 906 791 800 374 539 579 696 954 765 1240 436 589506 1036 950 723 1270 319 651 411 798 945 594 1000 411 649 612 975 878680 1140 364 653 501 872 829 692 1030 297 637 544 1014 911 746 1020 488631 609 869 787 544 1090 316 610 596 742 931 763 950 361 612 554 827 931583 1110 447 633 561 902 949 568 850 Sum 7626 12372 10886 17390 1797314024 20630 Average 381.30 618.60 544.30 869.50 898.65 701.20 1031.50Variance 4404.33 1743.94 4294.01 13456.37 7575.29 9217.75 13802.89

TABLE 11 Green Thunder 1 2 3 4 5 6 7 436 680 605 838 749 583 550 599 605767 712 884 598 730 487 631 581 818 833 534 730 448 614 457 820 830 752710 449 656 613 743 678 602 940 486 567 435 825 789 726 840 587 608 600892 875 452 940 463 594 482 740 881 632 900 535 653 548 820 746 672 830562 648 516 748 888 598 630 395 662 707 888 850 592 640 440 609 719 781894 691 760 593 627 449 842 821 608 890 472 608 596 756 747 664 910 458677 619 829 737 778 760 559 652 704 748 900 655 870 541 649 723 821 697658 760 480 602 529 879 745 831 920 567 604 569 814 829 654 750 493 573627 821 705 723 910 Sum 10050 12519 11846 16135 16078 13003 15970Average 502.50 625.95 592.30 806.75 803.90 650.15 798.50 Variance3651.11 1055.42 9477.91 2669.04 5328.83 7549.08 12918.68

TABLE 12 True Heart 1 2 3 4 5 6 7 495 850 588 795 732 1113 830 454 655738 860 756 1003 990 451 671 704 775 777 981 830 521 769 792 707 858 969800 645 743 522 672 810 958 740 484 586 740 745 783 993 1070 493 674 7131012 728 947 890 472 698 664 850 622 935 870 546 584 900 794 709 1027550 492 552 553 787 799 1116 880 342 739 492 795 955 944 870 552 774 893588 850 811 920 474 692 702 875 682 898 850 481 637 740 714 781 901 850441 798 527 694 745 888 760 628 579 787 891 751 891 980 566 598 593 779638 981 830 481 688 711 687 771 828 920 433 682 746 832 659 1269 840 499597 560 804 810 867 890 Sum 9950 13566 13665 15656 15216 19320 17160Average 497.50 678.30 683.25 782.80 760.80 966.00 858.00 Variance4575.21 6755.27 14006.0 8726.17 6240.06 11447.0 11174.74

Table 13 shows the results of a single-factor ANOVA testing showingdifferences in plant weight between varieties, locations, andinteractions between variety and location for data in Tables 10-12.Column 1 shows the source of variation, column 2 shows the sum ofsquares, column 3 shows the degrees of freedom, column 4 shows the meansquare, column 5 shows the F-value, column 6 shows the P-value, andcolumn 7 shows the F critical value. ANOVA results indicate significantdifferences in plant weight between varieties at harvest maturity.

TABLE 13 De- Source grees of of Varia- Sum of Free- Mean tion Squaresdom Square F-value P-value F crit Variety 288242.97 2 144121.4 18.90780.0000 3.02 Loca- 8417636.97 6 1402939.4 184.0562 0.0000 2.12 tionInter- 2156705.26 12 179725.44 23.5788 0.0000 1.78 action Within3041315.55 399 7622.34 Total 13903900.7 419

Tables 14 through 16 show the plant height in centimeters at harvestmaturity for Rio Bravo, (Table 14) Green Thunder, (Table 15) and TrueHeart (Table 16). The data were taken in 2008 TO 2010 at seven trials inWELLTON AND YUMA, ARIZONA, AND BARD AND SALINAS, CALIFORNIA for 20samples of each variety.

TABLE 14 Rio Bravo 1 2 3 4 5 6 7 25 33 33 39 36 34 34 28 36 34 38 36 3234 27 33 35 37 35 31 34 26 37 32 37 34 34 36 30 34 33 38 35 32 37 28 3235 38 35 33 33 29 33 33 37 35 33 32 28 35 33 34 36 35 32 27 33 34 36 3633 36 27 33 34 38 37 33 35 26 34 35 36 37 33 34 27 36 35 37 35 34 35 2834 33 36 34 29 35 29 35 34 38 36 30 33 27 34 34 37 35 33 35 29 33 34 3837 33 35 27 34 35 36 34 31 38 28 36 33 37 37 33 35 29 33 34 38 37 31 3526 32 33 37 37 32 34 Sum 551 680 676 742 714 649 692 Average 27.55 3433.8 37.1 35.7 32.45 34.6 Variance 1.6289 2.0000 0.8000 1.2526 1.16842.1553 2.2526

TABLE 15 Green Thunder 1 2 3 4 5 6 7 32 34 32 39 35 35 33 32 35 38 37 3235 33 32 36 33 39 36 33 34 31 34 33 37 37 33 32 31 35 37 38 34 33 37 3232 34 33 37 34 34 30 35 36 39 38 34 35 29 36 35 38 38 35 34 31 36 36 3734 34 33 28 36 36 36 34 35 32 33 35 34 38 37 35 33 32 35 34 37 36 34 3432 34 35 38 35 35 37 31 36 36 39 35 35 34 31 34 38 37 35 35 32 30 36 3736 38 36 32 33 34 38 38 35 36 31 32 35 36 37 35 35 36 31 36 35 39 35 3435 30 35 34 38 36 34 34 Sum 623 701 706 754 709 690 671 Average 31.1535.05 35.30 37.70 35.45 34.50 33.55 Variance 1.6079 0.6816 3.2737 0.95792.4711 0.7895 3.4184

TABLE 16 True Heart 1 2 3 4 5 6 7 30 33 37 39 37 35 31 30 35 36 39 37 3331 30 36 38 40 39 36 35 31 36 38 38 38 34 31 31 35 36 36 38 36 32 29 3639 37 37 35 33 28 34 36 38 37 34 32 32 34 36 38 39 33 32 28 33 36 38 3834 28 28 35 36 36 37 35 30 28 35 36 36 37 34 30 28 36 36 38 36 35 30 3131 33 38 37 37 33 32 36 37 38 37 35 33 30 35 37 37 38 33 31 29 34 36 3637 34 29 28 35 36 39 38 34 31 31 33 37 37 38 34 30 29 36 38 38 38 35 3030 34 36 37 37 34 32 Sum 593 694 735 752 750 686 621 Average 29.65 34.7036.75 37.60 37.50 34.30 31.05 Variance 1.9237 1.2737 0.9342 1.30530.5789 0.8526 2.4711

Table 17 shows the results of a single-factor ANOVA testing for plantheight between varieties, and within the varieties, Rio Bravo, GreenThunder, and True Heart, for data in Tables 14-16. Column 1 shows thesource of variation, column 2 shows the sum of squares, column 3 showsthe degrees of freedom, column 4 shows the mean square, column 5 showsthe F-value, column 6 shows the P-value, and column 7 shows the Fcritical value. ANOVA results indicate a significant difference in plantheight between varieties at harvest maturity.

TABLE 17 De- grees of Source of Sum of Free Mean Variation Squares domSquare F-value P-value F crit Variety 93.2333 2 46.6167 28.9653 0.00003.0183 Location 2404.9952 6 400.8325 249.0574 0.0000 2.1213 Interaction374.3333 12 31.1944 19.3827 0.0000 1.7765 Within 642.1500 399 1.6094Total 3514.7119 419

Tables 18 through 20 show the frame length in centimeters at harvestmaturity for Rio Bravo, (Table 18) Green Thunder, (Table 19) and TrueHeart (Table 20). The data were taken in 2008 TO 2010 at seven trials inWELLTON AND YUMA, ARIZONA, AND BARD AND SALINAS, CALIFORNIA for 20samples of each variety.

TABLE 18 Rio Bravo 1 2 3 4 5 6 7 26 29 31 34 32 31 33 27 31 30 35 34 3032 26 32 31 33 32 29 33 26 31 29 35 34 30 34 24 31 31 33 36 29 30 26 3029 34 33 29 34 24 29 31 35 32 29 32 27 31 30 33 34 30 34 26 32 29 33 3431 33 25 31 29 34 34 29 33 27 31 31 33 33 31 32 26 30 30 35 33 28 32 2632 30 35 33 28 29 25 30 31 34 35 28 27 26 30 29 33 35 30 29 26 31 30 3434 29 31 25 32 31 34 34 29 33 27 31 30 35 34 30 31 27 28 31 33 30 29 3126 30 31 35 32 27 33 Sum 518 612 604 680 668 586 636 Average 25.90 30.6030.20 34.00 33.40 29.30 31.80 Variance 0.8316 1.2000 0.6947 0.73681.8316 1.1684 3.5368

TABLE 19 Green Thunder 1 2 3 4 5 6 7 27 32 32 31 35 30 31 28 32 33 33 3432 30 29 30 32 32 35 33 32 28 31 29 33 36 30 29 29 32 34 34 35 33 32 2727 33 33 30 35 32 26 30 33 34 35 32 29 28 32 32 33 34 31 30 26 32 31 3334 32 30 27 33 33 33 36 31 28 27 33 33 32 36 32 31 26 31 29 34 34 31 3127 32 31 31 36 31 33 28 31 31 32 35 32 30 29 32 32 34 36 31 30 26 33 3331 34 33 29 27 31 33 33 34 32 27 28 32 30 33 35 33 31 29 32 31 34 36 3233 29 30 32 32 35 34 32 Sum 551 634 637 652 700 637 607 Average 27.5531.70 31.85 32.60 35.00 31.85 30.35 Variance 1.2079 0.9579 1.9237 1.41050.6316 1.0816 2.5553

TABLE 20 True Heart 1 2 3 4 5 6 7 27 30 35 34 36 31 26 26 31 35 36 35 3227 25 30 35 36 37 32 26 26 32 36 35 37 31 25 27 30 34 33 36 31 28 28 3133 36 36 31 28 26 30 35 34 36 32 26 27 31 35 36 35 32 27 26 30 36 36 3534 26 25 32 36 35 37 31 31 27 31 34 35 35 31 28 28 30 34 35 36 33 30 2525 28 36 36 37 31 28 31 34 34 37 33 28 26 30 35 36 37 33 29 27 31 35 3336 33 28 26 30 35 35 37 32 29 26 30 33 35 36 33 30 26 30 34 34 36 33 3127 31 34 34 37 30 30 Sum 529 609 694 698 724 639 561 Average 26.45 30.4534.70 34.90 36.20 31.95 28.05 Variance 0.8921 0.7868 0.8526 1.04210.5895 1.1026 3.1026

Table 21 shows the results of a two-factor ANOVA testing for frame leaflength between varieties, and within the varieties, Rio Bravo, GreenThunder, and True Heart, for data in Tables 18-20. Column 1 shows thesource of variation, column 2 shows the sum of squares, column 3 showsthe degrees of freedom, column 4 shows the mean square, column 5 showsthe F-value, column 6 shows the P-value, and column 7 shows the Fcritical value. ANOVA results indicate a significant difference in frameleaf length between varieties at harvest maturity.

TABLE 21 De- grees of Source of Sum of Free- Mean Variation Squares domSquare F-value P-value F crit Variety 87.6000 2 43.8000 32.6902 0.00003.0183 Location 2611.3571 6 435.2262 324.8321 0.0000 2.1213 Interaction532.5000 12 44.3750 33.1194 0.0000 1.7765 Within 534.6000 399 1.3398Total 3766.0571 419

Tables 22 through 24 shows the leaf width in centimeters at harvestmaturity for Rio Bravo, (Table 22) Green Thunder, (Table 23) and TrueHeart (Table 24). The data were taken in 2008 TO 2010 at seven trials inWELLTON AND YUMA, ARIZONA, AND BARD AND SALINAS, CALIFORNIA for 20samples of each variety.

TABLE 22 Rio Bravo 1 2 3 4 5 6 7 17 19 18 19 22 24 24 18 21 16 21 19 2121 16 20 17 21 21 22 24 16 19 18 21 22 22 23 16 20 18 21 23 20 21 16 2018 20 20 19 25 15 19 17 19 22 21 21 17 19 18 20 19 21 27 16 20 17 21 2221 24 16 19 18 20 22 22 24 17 20 17 21 22 21 24 18 19 18 20 21 17 23 1620 16 21 21 19 24 17 21 17 20 22 20 24 16 18 18 21 20 21 23 16 18 18 2120 21 22 18 19 18 19 20 19 23 16 20 17 19 22 20 22 18 19 18 20 19 20 2316 19 17 20 20 17 21 Sum 331 389 349 405 419 408 463 Average 16.55 19.4517.45 20.25 20.95 20.40 23.15 Variance 0.7868 0.6816 0.4711 0.61841.5237 2.7789 2.3447

TABLE 23 Green Thunder 1 2 3 4 5 6 7 18 19 19 19 17 20 19 17 20 18 20 1818 20 18 19 18 18 18 21 21 17 19 16 17 17 21 19 17 20 20 18 18 20 21 1716 18 19 19 18 18 16 20 19 19 17 19 19 17 18 18 19 17 17 22 17 19 18 1817 22 20 18 21 20 17 17 18 18 18 19 17 17 17 20 17 17 20 17 18 18 20 2016 18 19 18 18 18 20 18 18 18 19 17 21 23 18 19 19 20 17 20 22 17 19 1819 17 19 22 16 19 18 17 17 21 21 17 20 19 19 18 22 23 17 20 19 18 18 1919 18 19 17 18 18 20 20 Sum 343 384 366 367 349 394 406 Average 17.1519.20 18.30 18.35 17.45 19.70 20.30 Variance 0.5553 0.6947 1.0632 0.87110.2605 2.0105 2.5368

TABLE 24 True Heart 1 2 3 4 5 6 7 16 19 20 19 16 19 18 18 19 21 21 16 2120 17 19 19 19 18 22 19 17 18 18 20 17 19 20 16 19 23 20 17 20 18 17 1823 23 18 19 21 18 18 19 21 17 19 18 17 19 22 20 16 20 20 16 19 18 20 1819 20 16 19 17 19 17 20 21 18 20 17 22 16 20 18 17 19 19 20 18 20 19 1717 19 21 20 17 19 16 19 21 21 17 21 20 17 18 23 20 18 18 21 16 18 18 1918 21 18 17 19 17 19 18 18 18 18 18 17 20 18 19 20 17 19 19 22 17 21 2016 20 20 21 17 20 21 Sum 337 376 392 406 344 395 390 Average 16.85 18.8019.60 20.30 17.20 19.75 19.50 Variance 0.5553 0.3789 4.3579 1.27370.5895 1.1447 1.3158

Table 25 shows the results of a two-factor ANOVA testing for leaf widthbetween varieties, and within the varieties, Rio Bravo, Green Thunder,and True Heart, for data in Tables 22-24. Column 1 shows the source ofvariation, column 2 shows the sum of squares, column 3 shows the degreesof freedom, column 4 shows the mean square, column 5 shows the F-value,column 6 shows the P-value, and column 7 shows the F critical value.ANOVA results indicate a significant difference in leaf width betweenvarieties at harvest maturity.

Degrees Source of Sum of of Free- Mean Variation Squares dom SquareF-value P-value F crit Variety 96.1000 2 48.0500 37.6326 0.0000 3.0183Location 621.5571 6 103.5929 81.1337 0.0000 2.1213 Interaction 337.300012 28.1083 22.0144 0.0000 1.7765 Within 509.4500 399 1.2768 Total1564.4071 419

Tables 26 through 28 show the leaf index (calculated by dividing theleaf length by the leaf width) at harvest maturity for Rio Bravo, (Table26) Green Thunder, (Table 27) and True Heart (Table 28). The data weretaken in 2008 TO 2010 at seven trials in WELLTON AND YUMA, ARIZONA, ANDBARD AND SALINAS, CALIFORNIA for 20 samples of each variety.

TABLE 26 Rio Bravo 1 2 3 4 5 6 7 1.53 1.53 1.72 1.79 1.45 1.29 1.38 1.501.48 1.88 1.67 1.79 1.43 1.52 1.63 1.60 1.82 1.57 1.52 1.32 1.38 1.631.63 1.61 1.67 1.55 1.36 1.48 1.50 1.55 1.72 1.57 1.57 1.45 1.43 1.631.50 1.61 1.70 1.65 1.53 1.36 1.60 1.53 1.82 1.84 1.45 1.38 1.52 1.591.63 1.67 1.65 1.79 1.43 1.26 1.63 1.60 1.71 1.57 1.55 1.48 1.38 1.561.63 1.61 1.70 1.55 1.32 1.38 1.59 1.55 1.82 1.57 1.50 1.48 1.33 1.441.58 1.67 1.75 1.57 1.65 1.39 1.63 1.60 1.88 1.67 1.57 1.47 1.21 1.471.43 1.82 1.70 1.59 1.40 1.13 1.63 1.67 1.61 1.57 1.75 1.43 1.26 1.631.72 1.67 1.62 1.70 1.38 1.41 1.39 1.68 1.72 1.79 1.70 1.53 1.43 1.691.55 1.76 1.84 1.55 1.50 1.41 1.50 1.47 1.72 1.65 1.58 1.45 1.35 1.631.58 1.82 1.75 1.60 1.59 1.57 Sum 31.3598 31.5068 34.6716 33.639331.9716 28.8533 27.5648 Aver- 1.5680 1.5753 1.7336 1.6820 1.5986 1.44271.3782 age Vari- 0.0060 0.0056 0.0082 0.0080 0.0100 0.0081 0.0117 ance

TABLE 27 Green Thunder 1 2 3 4 5 6 7 1.50 1.68 1.68 1.63 2.06 1.50 1.631.65 1.60 1.83 1.65 1.89 1.78 1.50 1.61 1.58 1.78 1.78 1.94 1.57 1.521.65 1.63 1.81 1.94 2.12 1.43 1.53 1.71 1.60 1.70 1.89 1.94 1.65 1.521.69 1.69 1.83 1.74 1.58 1.94 1.78 1.63 1.50 1.74 1.79 2.06 1.68 1.531.65 1.78 1.78 1.74 2.00 1.82 1.36 1.53 1.68 1.72 1.83 2.00 1.45 1.501.50 1.57 1.65 1.94 2.12 1.72 1.56 1.50 1.74 1.94 1.88 2.12 1.60 1.821.53 1.55 1.71 1.89 1.89 1.55 1.55 1.69 1.78 1.63 1.72 2.00 1.72 1.651.56 1.72 1.72 1.68 2.06 1.52 1.30 1.61 1.68 1.68 1.70 2.12 1.55 1.361.53 1.74 1.83 1.63 2.00 1.74 1.32 1.69 1.63 1.83 1.94 2.00 1.52 1.291.65 1.60 1.58 1.74 1.94 1.50 1.35 1.71 1.60 1.63 1.89 2.00 1.68 1.741.61 1.58 1.88 1.78 1.94 1.70 1.60 Sum 32.1646 33.0799 34.8761 35.623140.1471 32.4810 30.0811 Aver- 1.6082 1.6540 1.7438 1.7812 2.0074 1.62401.5041 age Vari- 0.0053 0.0079 0.0085 0.0132 0.0055 0.0139 0.0210 ance

TABLE 28 True Heart 1 2 3 4 5 6 7 1.69 1.58 1.75 1.79 2.25 1.63 1.441.44 1.63 1.67 1.71 2.19 1.52 1.35 1.47 1.58 1.84 1.89 2.06 1.45 1.371.53 1.78 2.00 1.75 2.18 1.63 1.25 1.69 1.58 1.48 1.65 2.12 1.55 1.561.65 1.72 1.43 1.57 2.00 1.63 1.33 1.44 1.67 1.84 1.62 2.12 1.68 1.441.59 1.63 1.59 1.80 2.19 1.60 1.35 1.63 1.58 2.00 1.80 1.94 1.79 1.301.56 1.68 2.12 1.84 2.18 1.55 1.48 1.50 1.55 2.00 1.59 2.19 1.55 1.561.65 1.58 1.79 1.75 2.00 1.65 1.58 1.47 1.47 1.47 1.71 1.80 2.18 1.631.75 1.63 1.62 1.62 2.18 1.57 1.40 1.53 1.67 1.52 1.80 2.06 1.83 1.381.69 1.72 1.94 1.74 2.00 1.57 1.56 1.53 1.58 2.06 1.84 2.06 1.78 1.611.44 1.67 1.94 1.75 2.00 1.74 1.50 1.53 1.58 1.79 1.55 2.12 1.57 1.551.69 1.55 1.70 1.62 2.18 1.50 1.43 Sum 31.4620 32.4275 35.8009 34.478342.1589 32.4406 28.8331 Aver- 1.5731 1.6214 1.7900 1.7239 2.1079 1.62201.4417 age Vari- 0.0095 0.0053 0.0399 0.0105 0.0076 0.0101 0.0106 ance

Table 29 shows the results of a single-factor ANOVA testing for the leafindex between varieties, and within the varieties, Rio Bravo, GreenThunder, and True Heart, for data in Tables 26-28. Column 1 shows thesource of variation, column 2 shows the sum of squares, column 3 showsthe degrees of freedom, column 4 shows the mean square, column 5 showsthe F-value, column 6 shows the P-value, and column 7 shows the Fcritical value. ANOVA results indicate a significant difference in theleaf index between varieties at harvest maturity.

TABLE 29 Source of Sum of Degrees of Mean Variation Squares FreedomSquare F-value P-value F crit Variety 1.6253 2 0.8126 75.3459 0.00003.0183 Location 8.3222 6 1.3870 128.6010 0.0000 2.1213 Interaction2.0950 12 0.1746 16.1867 0.0000 1.7765 Within 4.3034 399 0.0108 Total16.3460 419

Tables 30 through 32 show the leaf area (calculated by multiplying theleaf length by the leaf width) at harvest maturity for Rio Bravo, (Table30) Green Thunder, (Table 31) and True Heart (Table 32). The data weretaken in 2008 TO 2010 at seven trials in WELLTON AND YUMA, ARIZONA, ANDBARD AND SALINAS, CALIFORNIA for 20 samples of each variety.

TABLE 30 Rio Bravo 1 2 3 4 5 6 7 442 551 558 646 704 744 792 486 651 480735 646 630 672 416 640 527 693 672 638 792 416 589 522 735 748 660 782384 620 558 693 828 580 630 416 600 522 680 660 551 850 360 551 527 665704 609 672 459 589 540 660 646 630 918 416 640 493 693 748 651 792 400589 522 680 748 638 792 459 620 527 693 726 651 768 468 570 540 700 693476 736 416 640 480 735 693 532 696 425 630 527 680 770 560 648 416 540522 693 700 630 667 416 558 540 714 680 609 682 450 608 558 646 680 551759 432 620 510 665 748 600 682 486 532 558 660 570 580 713 416 570 527700 640 459 693 Sum 8579 11908 10538 13766 14004 11979 14736 Average428.95 595.40 526.90 688.30 700.20 598.95 736.80 Variance 1022.781373.41 542.83 740.01 3201.12 4334.16 5477.01

TABLE 31 Green Thunder 1 2 3 4 5 6 7 486 608 608 589 595 600 589 476 640594 660 612 576 600 522 570 576 576 630 693 672 476 589 464 561 612 630551 493 640 680 612 630 660 672 432 432 594 627 570 630 576 416 600 627646 595 608 551 476 576 576 627 578 527 660 442 608 558 594 578 704 600486 693 660 561 612 558 504 486 627 561 544 612 640 527 442 620 493 612612 620 620 432 576 589 558 648 558 660 504 558 558 608 595 672 690 522608 608 680 612 620 660 442 627 594 589 578 627 638 432 589 594 561 578672 567 476 640 570 627 630 726 713 493 640 589 612 648 608 627 522 570544 576 630 680 640 Sum 9456 12173 11670 11963 12215 12555 12321 Average472.80 608.60 583.50 598.15 610.75 627.75 616.05 Variance 1088.481084.03 2456.47 1357.61 509.04 2867.04 3263.94

TABLE 32 True Heart 1 2 3 4 5 6 7 432 570 700 646 576 589 468 468 589735 756 560 672 540 425 570 665 684 666 704 494 442 576 648 700 629 589500 432 570 782 660 612 620 504 476 558 759 828 648 589 588 468 540 665714 612 608 468 459 589 770 720 560 640 540 416 570 648 720 630 646 520400 608 612 665 629 620 651 486 620 578 770 560 620 504 476 570 646 700648 660 570 425 425 532 756 720 629 589 448 589 714 714 629 693 560 442540 805 720 666 594 609 432 558 630 627 648 693 504 442 570 595 665 666576 522 468 540 561 700 648 627 600 442 570 646 748 612 693 620 432 620680 714 629 600 630 Sum 8911 11449 13595 14171 12457 12622 10952 Average445.55 572.45 679.75 708.5500 622.85 631.10 547.60 Variance 514.05624.99 4971.04 2105.31 1197.61 1737.25 2985.62

Table 33 shows the results of a two-factor ANOVA testing for the leafarea between varieties, and within the varieties, Rio Bravo, GreenThunder, and True Heart, for data in Tables 30-32. Column 1 shows thesource of variation, column 2 shows the sum of squares, column 3 showsthe degrees of freedom, column 4 shows the mean square, column 5 showsthe F-value, column 6 shows the P-value, and column 7 shows the Fcritical value. ANOVA results indicate a significant difference in leafarea between varieties at harvest maturity.

TABLE 33 Source of Sum of Degrees of Mean Variation Squares FreedomSquare F-value  P-value F crit Variety 35837.3190 2 17918.659 8.65960.0002 3.0183 Location 1832966.014 6 305494.34 147.6368 0.0000 2.1213Interaction 848011.1143 12 70667.593 34.1516 0.0000 1.7765 Within825622.6000 399 2069.2296 Total 3542437.047 419

Tables 34 through 36 show the core length in centimeters at harvestmaturity for Rio Bravo, (Table 34) Green Thunder, (Table 35) and TrueHeart (Table 36). The data were taken in 2008 TO 2010 at seven trials inWELLTON AND YUMA, ARIZONA, AND BARD AND SALINAS, CALIFORNIA for 20samples of each variety.

TABLE 34 Rio Bravo 1 2 3 4 5 6 7 3.8 5.6 6.0 9.0 5.9 8.0 8.1 3.2 6.1 6.08.3 6.9 6.8 8.1 4.0 5.2 4.3 7.9 6.0 7.1 7.0 3.6 5.4 5.4 8.5 5.4 7.9 10.03.5 5.7 5.7 7.9 5.4 6.2 8.0 3.2 6.0 6.0 7.9 5.2 7.3 8.2 3.5 6.0 4.8 8.76.2 9.0 9.1 3.6 5.9 5.6 8.1 7.1 9.0 8.2 4.0 5.8 6.1 8.3 6.5 9.0 8.6 3.96.1 5.9 8.8 6.6 9.0 9.2 3.4 5.5 5.3 7.9 7.1 8.0 8.1 3.3 5.6 5.4 8.2 6.49.0 6.0 3.7 5.9 5.3 8.4 6.0 6.0 7.2 3.7 6.1 5.9 8.1 7.0 7.0 8.0 3.8 6.05.0 8.6 4.5 6.5 8.1 4.0 5.6 5.6 8.6 6.8 7.0 7.5 3.8 6.0 5.9 8.4 5.9 6.07.0 3.5 6.3 6.1 8.1 6.8 7.0 8.4 3.6 6.0 5.4 8.3 7.5 6.1 8.5 3.4 5.8 4.98.4 7.1 5.6 8.4 Sum 72.5 116.6 110.6 166.4 126.3 147.5 161.7 Average3.6250 5.8300 5.5300 8.3200 6.3150 7.3750 8.0850 Variance 0.0641 0.07690.2443 0.1006 0.6013 1.3578 0.7603

TABLE 35 Green Thunder 1 2 3 4 5 6 7 4.4 5.0 5.5 9.1 6.0 6.7 6.0 4.1 5.85.6 8.2 4.8 7.0 7.5 3.7 4.9 3.6 8.8 5.9 6.5 7.5 3.7 5.9 3.2 7.9 6.1 7.76.5 3.4 5.2 5.2 8.2 6.2 7.5 8.5 4.2 4.2 5.2 5.2 6.0 5.3 8.5 4.2 5.5 5.38.4 5.4 7.2 8.0 3.6 4.9 4.5 8.6 6.0 7.5 8.5 4.5 5.7 5.1 8.1 5.0 7.0 7.53.3 5.1 5.8 6.4 6.1 6.5 6.0 3.6 5.3 5.6 7.7 6.0 8.0 7.0 3.2 5.8 4.1 8.84.6 8.0 9.0 4.2 4.9 4.3 8.9 5.9 6.0 6.0 4.6 6.0 3.8 7.8 6.0 7.0 6.3 4.16.2 4.1 8.1 5.7 8.0 6.5 3.2 5.8 5.7 6.9 5.0 6.6 6.7 4.0 5.7 5.2 8.7 4.67.0 7.7 4.3 5.0 5.3 8.0 5.8 6.0 8.0 4.3 5.8 4.9 7.9 5.1 6.2 8.5 3.7 5.35.5 8.4 5.9 6.9 7.5 Sum 78.3 109 97.5 160.9 111.4 141.8 145.2 Average3.9150 5.4500 4.8750 8.0450 5.5700 7.0900 7.2600 Variance 0.1929 0.17320.5978 0.6552 0.2917 0.5009 0.9457

TABLE 36 True Heart 1 2 3 4 5 6 7 3.2 6.1 5.1 6.7 5.5 8.2 6.5 3.6 5.75.7 7.2 5.7 6.2 7.8 3.6 6.1 5.6 6.3 5.6 7.1 7.2 3.9 6.8 5.2 7.7 6.3 7.05.0 4.7 4.6 5.0 7.1 6.7 7.1 5.7 4.0 5.4 4.9 6.1 5.4 5.9 7.3 4.8 6.7 5.17.5 5.8 7.2 8.0 3.1 6.1 5.6 7.1 5.7 6.2 8.0 3.5 6.0 5.5 6.5 5.5 6.1 5.83.8 6.4 5.1 6.8 5.3 5.6 6.0 4.0 5.6 5.6 6.0 5.5 6.4 6.8 3.5 6.3 5.3 7.25.2 6.5 5.2 3.8 3.8 6.2 5.3 7.0 5.0 7.0 3.2 6.1 5.4 6.8 6.2 5.2 6.3 4.65.6 5.6 6.7 6.1 5.8 8.5 4.4 4.9 4.9 6.3 5.4 7.9 8.0 3.6 6.1 5.1 6.7 5.56.2 8.2 3.3 5.6 5.3 7.2 6.4 6.6 7.2 3.8 5.9 5.7 7.7 5.4 6.4 6.7 4.4 6.75.7 6.9 5.5 5.8 7.6 Sum 76.8 118.9 106.7 137.5 113.7 130.4 138.5 Average3.8400 5.9450 5.3350 6.8750 5.6850 6.5200 6.9250 Variance 0.2625 0.31630.0761 0.2346 0.1919 0.5712 1.0567

Table 37 shows the results of a two-factor ANOVA testing for the corelength between varieties, and within the varieties, Rio Bravo, GreenThunder, and True Heart, for data in Tables 34-36. Column 1 shows thesource of variation, column 2 shows the sum of squares, column 3 showsthe degrees of freedom, column 4 shows the mean square, column 5 showsthe F-value, column 6 shows the P-value, and column 7 shows the Fcritical value. ANOVA results indicate a significant difference in corelength between varieties at harvest maturity.

TABLE 37 De- grees of Source of Sum of Free- Mean Variation Squares domSquare F-value P-value F crit Variety 23.8800 2 11.9400 27.0428 0.00003.0183 Location 689.9235 6 114.9872 260.4327 0.0000 2.1213 Interaction36.0570 12 3.0047 6.8054 0.0000 1.7765 Within 176.1680 399 0.4415 Total926.0285 419

Tables 38 through 40 show the core diameter in centimeters at harvestmaturity for Rio Bravo, (Table 38) Green Thunder, (Table 39) and TrueHeart (Table 40). The data were taken in 2008 TO 2010 at seven trials inWELLTON AND YUMA, ARIZONA, AND BARD AND SALINAS, CALIFORNIA for 20samples of each variety.

TABLE 38 Rio Bravo 1 2 3 4 5 6 7 3.0 3.2 3.2 3.8 3.5 3.5 4.0 2.8 3.0 3.23.3 3.8 3.4 4.0 3.0 3.0 3.0 4.0 3.5 3.3 4.1 2.7 3.1 3.1 4.0 3.5 3.5 3.83.0 3.3 3.0 3.5 3.8 3.2 3.9 3.0 3.2 3.0 3.5 3.9 3.5 4.5 3.0 3.0 3.1 3.84.0 3.5 4.4 2.9 3.0 3.2 3.9 3.5 3.8 3.8 3.0 3.2 3.0 3.4 4.0 3.9 4.2 3.13.1 3.1 3.6 3.7 3.9 3.7 3.0 3.1 3.0 3.7 3.7 3.9 4.3 2.9 3.3 3.1 4.0 3.83.5 4.2 3.1 3.2 3.1 3.7 3.7 3.6 5.2 3.0 3.1 3.2 3.9 3.6 3.5 4.3 3.2 3.03.0 3.9 3.8 4.0 4.4 3.2 3.2 3.1 3.7 3.7 4.0 4.5 3.4 3.1 3.2 3.9 3.9 3.74.0 2.9 3.0 3.1 4.0 3.9 3.9 4.1 2.8 3.3 3.0 3.8 3.9 3.5 4.3 2.8 3.1 3.33.8 3.9 3.5 4.0 Sum 59.8 62.5 62 75.2 75.1 72.6 83.7 Aver- 2.9900 3.12503.1000 3.7600 3.7550 3.6300 4.1850 age Vari- 0.0262 0.0114 0.0084 0.04360.0279 0.0569 0.1119 ance

TABLE 39 Green Thunder 1 2 3 4 5 6 7 3.1 3.6 4.0 3.8 3.2 3.7 3.5 3.3 3.43.0 4.0 3.3 3.5 4.0 3.4 3.2 3.5 4.0 3.8 3.5 4.0 3.5 3.4 3.0 3.9 3.2 3.84.3 3.3 3.7 3.4 4.1 3.5 3.8 4.5 3.0 3.0 3.7 3.0 3.9 3.6 3.8 3.6 3.8 3.94.0 3.7 3.8 4.0 3.0 3.3 3.5 4.1 3.6 3.8 4.5 3.2 3.8 3.6 4.1 3.8 3.8 4.33.4 3.3 3.2 3.8 3.6 3.4 4.3 3.3 3.5 3.2 3.8 3.6 3.8 4.0 3.0 3.6 3.8 3.83.5 3.9 4.5 3.1 3.3 3.6 4.0 3.5 3.5 4.5 3.6 3.7 3.5 3.9 3.8 3.5 5.0 3.23.2 3.4 3.9 3.5 3.5 4.4 3.1 3.5 3.6 3.8 3.5 3.7 4.3 3.4 3.6 3.6 4.0 3.53.5 4.5 3.7 3.4 3.7 4.0 3.5 3.2 4.3 3.2 3.3 3.8 3.8 3.2 3.6 4.2 3.4 3.83.5 3.9 3.2 3.7 4.0 Sum 65.8 70.1 69.8 78.6 70.1 72.8 85.6 Aver- 3.29003.5050 3.4900 3.9300 3.5050 3.6400 4.2800 age Vari- 0.0441 0.0416 0.08520.0117 0.0394 0.0331 0.0964 ance

TABLE 40 True Heart 1 2 3 4 5 6 7 3.4 3.6 4.0 3.6 3.2 3.8 4.5 3.3 3.83.9 4.0 3.3 3.5 4.2 3.2 3.5 3.9 3.8 3.8 3.6 4.0 3.3 3.9 3.8 3.9 3.2 3.84.0 3.5 3.6 3.6 3.8 3.5 4.0 4.0 3.5 3.2 3.3 4.0 3.6 3.1 4.0 3.2 3.9 3.63.8 3.7 3.7 4.5 3.0 3.8 3.9 4.0 3.6 3.4 4.0 3.2 3.8 3.4 3.9 3.8 3.1 4.13.4 3.7 3.8 3.8 3.6 3.6 4.0 3.1 3.4 3.8 3.6 3.6 3.7 4.1 3.2 3.7 4.0 3.73.5 3.8 4.2 3.5 3.5 3.5 3.8 4.0 3.5 3.7 3.4 3.6 3.8 4.0 3.8 3.6 4.2 3.33.8 3.6 3.7 3.5 3.7 4.5 3.3 3.6 3.7 3.8 3.5 3.7 4.8 3.5 3.7 3.5 3.8 3.53.7 4.0 3.4 3.3 3.7 3.9 3.5 3.8 4.6 3.3 3.9 3.8 4.0 3.2 3.8 4.5 3.4 4.03.8 3.9 3.2 3.6 4.5 Sum 66.4 73.3 74.7 77 70.1 72.7 84.9 Aver- 3.32003.6650 3.7350 3.8500 3.5050 3.6350 4.2450 age Vari- 0.0196 0.0445 0.03500.0174 0.0394 0.0498 0.0647 ance

Table 41 shows the results of a single-factor ANOVA testing for the corediameter between varieties, and within the varieties, Rio Bravo, GreenThunder, and True Heart, for data in Tables 38-40. Column 1 shows thesource of variation, column 2 shows the sum of squares, column 3 showsthe degrees of freedom, column 4 shows the mean square, column 5 showsthe F-value, column 6 shows the P-value, and column 7 shows the Fcritical value. ANOVA results indicate a significant difference in corediameter between varieties at harvest maturity.

TABLE 41 Source of Sum of Degrees of Mean Variation Squares FreedomSquare F-value P-value F crit Variety 3.1299 2 1.5649 36.1870 0.00003.0183 Location 40.5796 6 6.7633 156.3920 0.0000 2.1213 Interaction6.5978 12 0.5498 12.7138 0.0000 1.7765 Within 17.2550 399 0.0432 Total67.5623 419

DEPOSIT INFORMATION

A deposit of the lettuce cultivar seed of this invention is maintainedby Syngenta Participations A.G, Inc., having an address at WerkRosental, Schwarzwaldallee 215, CH-4058 Basel. A deposit of the cultivarRio Bravo will be made with the American Type Culture Collection (ATCC),Manassas, Va. 20110, and an ATCC accession number will be given afterseeds deposited

Access to this deposit will be available during the pendency of thisapplication to persons determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. Upon allowance of any claims in this application, all restrictionson the availability to the public of the variety will be irrevocablyremoved by affording access to a deposit of at least 2,500 seeds of thesame variety with the American Type Culture Collection, Manassas, Va.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions, and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

1. A seed of lettuce cultivar Rio Bravo, wherein a representative sample of seed of said cultivar was deposited under ATCC Accession No. PTA-11834.
 2. A lettuce plant, or a part thereof, produced by growing the seed of claim
 1. 3. A tissue culture produced from protoplasts or cells from the plant of claim 2, wherein said cells or protoplasts are produced from a plant part selected from the group consisting of leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell root, root tip, pistil, anther, ovule, flower, shoot, stem, seed, and petiole.
 4. A lettuce plant regenerated from the tissue culture of claim 3, wherein the plant has all of the morphological and physiological characteristics of cultivar Rio Bravo.
 5. A method for producing a lettuce seed comprising crossing two lettuce plants and harvesting the resultant lettuce seed, wherein at least one lettuce plant is the lettuce plant of claim
 2. 6. A lettuce seed produced by the method of claim
 5. 7. A lettuce plant, or a part thereof, produced by growing said seed of claim
 6. 8. The method of claim 5, wherein at least one of said lettuce plants is transgenic.
 9. A method of producing a male sterile lettuce plant, wherein the method comprises introducing a nucleic acid molecule that confers male sterility into the lettuce plant of claim
 2. 10. A male sterile lettuce plant produced by the method of claim
 9. 11. A method of producing an herbicide resistant lettuce plant, wherein said method comprises introducing a gene conferring herbicide resistance into the plant of claim 2, wherein the gene is selected from the group consisting of glyphosate, sulfonylurea, imidazolinone, dicamba, glufosinate, phenoxy proprionic acid, L-phosphinothricin, cyclohexone, cyclohexanedione, triazine, and benzonitrile.
 12. An herbicide resistant lettuce plant produced by the method of claim
 11. 13. A method of producing a pest or insect resistant lettuce plant, wherein said method comprises introducing a gene conferring pest or insect resistance into the plant of claim
 2. 14. A pest or insect resistant lettuce plant produced by the method of claim
 13. 15. The lettuce plant of claim 14, wherein the gene encodes a Bacillus thuringiensis endotoxin.
 16. A method of producing a disease resistant lettuce plant, wherein said method comprises introducing a gene conferring disease resistance into the plant of claim
 2. 17. A disease resistant lettuce plant produced by the method of claim
 16. 18. A method of producing a lettuce plant with a value-added trait, wherein said method comprises introducing a gene conferring a value-added trait into the plant of claim 2, where said gene encodes a protein selected from the group consisting of a ferritin, a nitrate reductase, and a monellin.
 19. A lettuce plant with a value-added trait produced by the method of claim
 18. 20. A method of introducing a desired trait into lettuce cultivar Rio Bravo wherein the method comprises: (a) crossing a Rio Bravo plant, wherein a representative sample of seed was deposited under ATCC Accession No. PTA-11834, with a plant of another lettuce cultivar that comprises a desired trait to produce progeny plants wherein the desired trait is selected from the group consisting of male sterility, herbicide resistance, insect or pest resistance, modified bolting and resistance to bacterial disease, fungal disease or viral disease; (b) selecting one or more progeny plants that have the desired trait to produce selected progeny plants; (c) crossing the selected progeny plants with the Rio Bravo plant to produce backcross progeny plants; (d) selecting for backcross progeny plants that have the desired trait and all of the physiological and morphological characteristics of lettuce cultivar Rio Bravo listed in Table 1; and (e) repeating steps (c) and (d) two or more times in succession to produce selected third or higher backcross progeny plants that comprise the desired trait and all of the physiological and morphological characteristics of lettuce cultivar Rio Bravo listed in Table
 1. 21. A lettuce plant produced by the method of claim 20, wherein the plant has the desired trait.
 22. The lettuce plant of claim 21, wherein the desired trait is herbicide resistance and the resistance is conferred to an herbicide selected from the group consisting of glyphosate, sulfonylurea, imidazolinone, dicamba, glufosinate, phenoxy proprionic acid, L-phosphinothricin, cyclohexone, cyclohexanedione, triazine, and benzonitrile.
 23. The lettuce plant of claim 21, wherein the desired trait is insect or pest resistance and the insect or pest resistance is conferred by a transgene encoding a Bacillus thuringiensis endotoxin. 